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US6270993B1 - VEGF-binding polypeptide - Google Patents

VEGF-binding polypeptide Download PDF

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US6270993B1
US6270993B1 US09/051,363 US5136398A US6270993B1 US 6270993 B1 US6270993 B1 US 6270993B1 US 5136398 A US5136398 A US 5136398A US 6270993 B1 US6270993 B1 US 6270993B1
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dna
vegf
immunoglobulin
thr
flt
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Masabumi Shibuya
Masaji Okamoto
Mikio Niwa
Tomoe Matsumoto
Makoto Asano
Tosiaki Segawa
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Toagosei Co Ltd
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Toagosei Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to polypeptides which are useful as neovascularization inhibitors, and a method of producing them.
  • pathological neovascularization is closely related to the symptoms or causes of certain diseases. Solid tumors are representative of such diseases. For the growth of tumor tissue beyond the diameter of 1 to 2 mm, newly formed blood vessels need to extend from the existing blood vessels to reach the tumor tissue (J. Folkman, J. Natl. Cancer Inst., 82:4 (1990)). When the blood vessel reaches the tumor tissue, its growth is explosively accelerated. Diabetic retinopathy is accompanied by pathological neovascularization of the retina, which often leads to the loss of eyesight.
  • pathological neovascularization is also seen in such diseases as chronic rheumatoid arthritis, psoriasis, hemangioma, scleroderma, and neovascular glaucomas, and it is considered to be one of the main symptoms (J. Folkman and N. Engle, J. Med., 320:1211 (1989)). Therefore, it may be possible to use substances that inhibit neovascularization for the treatment of tumors and other diseases mentioned above.
  • Vascular endothelial cells are the cells that constitute the innermost layer of the blood vessel. Neovascularization occurs when vascular endothelial cells proliferate upon stimulation by growth factors, physiologically active substances, or mechanical damages.
  • growth factors that can directly or indirectly stimulate the proliferation of vascular endothelial cells include bFGF (basic Fibroblast Growth Factor), aFGF (acidic Fibroblast Growth Factor), VEGF (Vascular Endothelial cell Growth Factor), PD-ECGF (Platelet-Derived Endothelial Cell Growth Factor), TNF- ⁇ (Tumor Necrosis Factor- ⁇ ), PDGF (Platelet-Derived Growth Factor), EGF (Epidermal Growth Factor), TGF- ⁇ (Transforming Growth Factor- ⁇ ), and HGF (Hepatocyte Growth Factor) (L.
  • bFGF basic Fibroblast Growth Factor
  • aFGF acidic Fibroblast Growth Factor
  • VEGF Vascular Endotheli
  • VEGF can be distinguished from the other growth factors by the fact that its action is very specific to vascular endothelial cells. In other words, the VEGF receptor is found in very few cells other than vascular endothelial cells.
  • VEGF is a glycoprotein whose molecular weight is 40,000-45,000, and exists as a dimer (P. W. Leung et al., Science, 246:1306 (1989), P. J. Keck et al., Science, 246: 1319 (1989)). VEGF acts, by binding to the VEGF receptor, to promote cell proliferation and enhance membrane permeability.
  • Anti-VEGF neutralizing antibodies exhibit anti-tumor activities in tumor-bearing mice (K. J. Kim et al., Nature, 362:841 (1993), S. Kondo et al., Biochem. Biophys. Res. Commun., 194:1234 (1993)). From these facts, it is considered that VEGF secreted by tumor cells plays a major role in neoplastic neovascularization.
  • FLT In humans there are two known VEGF receptors, FLT (M. Shibuya et al., Oncogene, 5:519 (1990)) and KDR (B. I. Terman et al., Biochem. Biophys. Res. Commun., 187:1579 (1992)).
  • FLT-1 The extracellular domain of FLT (also known in the art as FLT-1) has seven immunoglobulin-like domains as shown in FIG. 1 (C. DeVries et al., Science, 255:989 (1992)).
  • FLT a cDNA of a soluble-type receptor has been cloned (R. L. Kendal and K. A. Thomas, Proc. Natl. Acad. Sci.
  • the polypeptide encoded by this cDNA corresponds to the first through sixth immunoglobulin-like domains of the seven immunoglobulin-like domains of the FLT extracellular domain, and it inhibited the VEGF activities by binding to VEGF with an affinity comparable to that of the full-length FLT.
  • KDR it is also known that the genetically engineered first through sixth immunoglobulin-like domains of the extracellular domain bind to VEGF (R. L. Kendal et al., Biochem. Biophys. Res. Commun., 201:326(1994)).
  • mouse anti-VEGF neutralizing antibodies exhibit antitumor activity, they are expected to be useful as anti-cancer agents.
  • human antibodies against the mouse antibody may be produced, which could lead to neutralization of the mouse antibody or might cause anaphylactic shock.
  • the extracellular domain of the VEGF receptor specifically binds to VEGF with high affinity, thereby inhibiting the VEGF activity, it can be considered useful as an inhibitor against neovascularization.
  • the possibility of antibody production in a human recipient is expected to be low because it is a polypeptide of human origin.
  • soluble CD4 which is a receptor for HIV, is 15 minutes (D. J. Capon et al., Nature, 337:525 (1989)), and that of interferon ⁇ is 30 minutes (I. Rutenfranz and H. Kirchner, J. Interferon Res., 8:573 (1988)).
  • a fusion polypeptide genetically engineered by combining the polypeptide of interest with a molecule having a long plasma half-life such as an antibody molecule.
  • the plasma half-life was increased from 15 min to 48 hr when it was chimerilized with the Fc domain of IgG1 (D. J. Capon et al., Nature, 337:525 (1989)).
  • Such a fusion polypeptide with the Fc domain of an antibody is also expected to provide an effect to induce the effector functions that the antibody possesses, i.e., complement-dependent cytotoxicity (D. B.
  • a fusion polypeptide constructed with an antibody it is desirable to select a polypeptide with a low molecular weight as a starting material because the molecular weight increases through the fusion. This is because, if a high molecular weight polypeptide is used, the molecular weight of the corresponding DNA is also high, which is to be handled by gene manipulation upon production of the recombinant host that produces the fusion polypeptide.
  • the larger the molecular weight of the DNA to be introduced the less efficient the transfection of the host becomes, thereby reducing the productivity of the recombinant host.
  • the larger the molecular weight of the recombinant polypeptide to be produced the smaller the amount of product tends to be.
  • large molecular weight polypeptides have been reported to show a poor infiltration capability into the diseased area (D. M. Lane et al., Br. J. Cancer, 70:521 (1994)).
  • polypeptides which can inhibit neovascularization by specifically inhibiting VEGF, and particularly those which are contained in the extracellular domain of the VEGF receptor.
  • polypeptides containing immunoglobulin-like domain 1 and immunoglobulin-like domain 2 of the extracellular domain of FLT can inhibit the VEGF activities by specifically binding to VEGF with high affinity.
  • polypeptides used herein means molecules constituted by amino acids that are covalently bound to each other via peptide bonds, and the lengths of the molecules are not limited.
  • polypeptides of the present invention include, in addition to the ones consisting of immunoglobulin-like domain 1 and immunoglobulin-like domain 2 of the extracellular domain of FLT, those which contain other domains.
  • they include the polypeptides containing immunoglobulin-like domains 1 through 4 and those containing immunoglobulin-like domains 1 through 5.
  • each domain is defined herein as the one that contains the amino acid sequence designated by the following amino acid residue numbers.
  • the amino acid residue numbers are the same as those shown in SEQ ID NO:1. Namely, they correspond to the residue numbers counted from the amino-terminal “Ser” of the mature FLT, which is position 1 in SEQ ID NO:1.
  • the present invention includes the polypeptides constructed by fusing the above extracellular domain of FLT with another protein (such as the Fc domain of immunoglobulins).
  • RNA is extracted by the acid phenol method (P. Chomzynski and N. Sacchi, Anal. Biochem., 162:156 (1987)) from the cultured human vascular endothelial cells, such as human umbilical chord-derived vascular endothelial cells (commercially available from Iwaki Glass, Morinaga Dairy Products, or Kurabo), and purified into a poly A + RNA using an oligo dT cellulose.
  • a single-stranded or double-stranded cDNA is synthesized using this RNA as a template, reverse transcriptase, and the oligo dT (12-16) primer.
  • the poly A + RNA and the cDNA can be prepared in accordance with “Molecular Cloning” (by J. Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Alternatively, the commercially available poly A + RNA preparation reagents (oligotex-dT30, Takara) or cDNA synthesis kit (Pharmacia Biosystem) can be used. If an flt cDNA has been already cloned from a cDNA library, the DNA corresponding to the region to be expressed can be isolated by digestion with appropriate restriction enzymes and introduced directly into an expression vector.
  • a desired part of the DNA can be amplified by PCR using the cDNA obtained above as the template (refer to “PCR protocols”, Academic Press Inc., 1990).
  • the following primers may be used.
  • the primer DNA can be synthesized with a DNA synthesizer (Applied Biosystems, Japan Millipore Ltd., etc.) or custom-made (Sawadee Technology).
  • a DNA synthesizer Applied Biosystems, Japan Millipore Ltd., etc.
  • custom-made Sawadee Technology
  • Upstream primer 5′-N (3-5) X (6) CGTCGCGCTCACCATGGTCAG-3′ (SEQ ID NO:2) downstream primer: 5′-N (3-5) Y (6) TTATTCGTAAATCTGGGGTTTCAC-3′ (SEQ ID NO:3).
  • N stands for A, C, G, or T
  • X or Y stands for a restriction enzyme recognition sequence
  • the numeral in the parentheses indicates the number of nucleotides.
  • N (3-5) means that there are 3 to 5 nucleotides of A, C, G, and T
  • X (6) or Y (6) indicates the recognition site for a 6-base cutter restriction enzyme. It is desirable to choose sequences that are found in neither the DNA fragment to be amplified nor the vector to which the fragment will be inserted as the restriction enzyme recognition sequences in the above.
  • the downstream primers can be appropriately designed to amplify the DNA fragments encoding the desired carboxy-termini.
  • the polypeptide-coding sequences When inserted into an expression vector, it should be noted that the polypeptide-coding sequences must be placed under the control of a promoter sequence. Parts of the primer sequences which correspond to the fit DNA sequence do not need to be exactly limited to 21 bases, but could be about 17-25 bases.
  • the condition for PCR can be a standard one as described in the “PCR Protocols” above, the reaction may be optimized to achieve a better efficiency by appropriately changing various parameters (e.g., Mg 2+ concentration, annealing temperature, extension time, the number of cycles, etc.), since the reaction proceeds differently depending on the template quantity and the primer sequences.
  • Pfu polymerase As the DNA polymerase used for PCR, Pfu polymerase (Stratagene), which possesses a proofreading (3′ exonuclease) activity, or Taq polymerase supplemented with Pfu polymerase will provide a better fidelity during the PCR amplification than Taq polymerase alone (W. M. Barnes, Proc. Natl. Acad. Sci. U.S.A., 91:2216 (1994)).
  • sequence of the DNA fragment to be amplified by PCR is known in this case, whether the desired DNA fragment has been obtained can be determined by, after amplification, confirming its size by agarose gel electrophoresis, recovering the fragment from the gel, digesting it with appropriate restriction enzymes, and examining the resulting electrophoresis pattern.
  • Agarose gel electrophoresis, recovering of DNA fragments from the gel, and restriction enzyme digestions can be done according to the “Molecular Cloning” above.
  • a commercial kit which utilizes glass beads for example, Prep-A-Gene, BIORAD) can be used for recovering DNA from a gel.
  • the recovered DNA fragment is digested with the restriction enzymes capable of cutting X (6) and Y (6) on both ends, deproteinated by the phenol treatment, ethanol-precipitated, and resuspended in an appropriate buffer, such as TE (10 mM Tris-HCl (pH 7.5)/1 mM EDTA).
  • an appropriate buffer such as TE (10 mM Tris-HCl (pH 7.5)/1 mM EDTA).
  • TE mM Tris-HCl (pH 7.5)/1 mM EDTA.
  • the cloning sites of an appropriate expression vector are digested with the restriction enzymes capable of cleaving X (6) and Y (6), agarose gel electrophoresis is performed, and the vector DNA is recovered. Through this procedure, a small fragment between the X (6) and Y (6) recognition sites is eliminated.
  • the ligation product is then added to competent E. coli cells, the transformation of the cells is performed, and transformants are screened by the antibiotic resistance on a culture medium containing the antibiotic corresponding to the selection marker (e.g., ampicillin resistance, kanamycin resistance, etc.) encoded by the vector.
  • the selection marker e.g., ampicillin resistance, kanamycin resistance, etc.
  • the recombinant expression vector, to which the DNA fragment has been inserted can be selected by examining the restriction enzyme digestion patterns of the plasmids in the antibiotic-resistant transformants. Alternatively, whether a transformant is a recombinant or not can be examined by performing a PCR reaction on the whole bacteria as the template, using the same set of primers as used to amplify the insert DNA, and detecting the presence or absence of the amplified target fragment. These series of procedures to obtain recombinant E. coli can be performed according to the “Molecular Cloning” above.
  • a variety of hosts can be used in order to produce the polypeptides of the present invention.
  • Gram negative and Gram positive bacteria such as Escherichia coli , bacteria belonging to the genus Pseudomonas, Bacillus subtilis, Bacillus brevis, Bacillus liqueniformis , and Bacillus thuringenesis ; yeast such as Pichia pastoris, Schizosaccharomyces pombe , and Saccharoimyces cerevisiae ; Eumycetes such as those belonging to the genus Aspergillus; insect cells such as Sf9 (derived from Spodoptera frugiperda ), Sf21, TN5 (derived from Trichoplusia ni ), and BN4 (derived from Bombyx moli ); and mammalian cells such as CHO (derived from the Chinese hamster ovary) and COS (derived from the monkey kidney).
  • the vector can be selected based on the suitability to the host cells.
  • the final transformants may be easily obtained by producing the recombinant DNA first in E. coli using a shuttle vector functioning in the host to be used for production of the polypeptide of the present invention and E. coli .
  • the transformations method used for obtaining the recombinant host that produces the polypeptide of the present invention include the competent cell method for E. coli ; the competent cell method (K. Bott and G. A. Wilson, J. Bacteriol., 94:562 (1967)) and the protoplast method (M. Mandel and A. Higa, J. Mol. Biol., 53:159 (1970)) for bacteria belonging to the genus Bacillus; the protoplast method (M.
  • the DNA encoding the region to be expressed can be inserted into the plasmid or viral DNA capable of replicating in the host downstream from a strong promoter that functions in the host. If the gene to be expressed is missing the translation initiation codon, it needs to be added.
  • the ribosome binding sequence J. R. MacLaughlin et al., J. Biol. Chem., 256:11283 (1981) is necessary.
  • the polypeptide may be recovered from the body fluid of the silkworms more efficiently by constructing a recombinant virus from BmNPV, which is a silkworm virus, and inoculating it into silkworms (H. Kawai and Y. Shimomura, Bioindustry, 8:39 (1991)).
  • the recombinant polypeptide may be obtained by transplanting the mouse myeloma cells transformed with a recombinant pSV vector into the abdominal cavity of a SCID or nude mouse and recovering the polypeptide from the abdominal fluid of the mouse. It may also be possible to use as hosts transgenic animals (G. Wright et al., Bio/Technology, 9:830 (1991)) or transgenic plants (M. Owen et al., Bio/Technology, 10:790 (1992)) constructed with the DNA of the present invention.
  • the signal peptide coding region of FLT can be used as it is if a eukaryotic cell is used as the host. If a bacterium is used as the host, the DNA encoding the signal peptide of a host's secreted polypeptide may be utilized. For example, for use in E. coli host cells, signal peptide-encoding DNA can be derived from E.
  • signal peptide-encoding DNA can be derived from Bacillus genes encoding amylases, alkaline phosphatases, and serine proteases, whose nucleotide sequences are known. If intracellular expression is desired, the signal peptide coding region except the initiation codon can be excluded. When an exogenous polypeptide is expressed at a high level in bacterial cells, inclusion bodies are often formed.
  • the inclusion bodies are dissolved in an 8 M urea solution, diluted to a polypeptide concentration of several ⁇ g/ml, and then dialyzed to gradually remove the urea, thereby recovering several percents of activity of the polypeptide. It is also possible to suppress the formation of inclusion bodies by concurrently expressing E. coli thioredoxin at a high level in the bacterial cells.
  • the polypeptide of the present invention produced by the methods as described above can be purified through usual biochemical means, including, for example, ammonium sulfate precipitation, ion exchange chromatography, gel filtration, and hydrophobic chromatography. Since the polypeptide of the present invention has affinity to heparin, the affinity chromatography with heparin resin can be utilized. When it is produced as a fusion polypeptide with another polypeptide, it can be purified by taking advantage of the properties possessed by partner polypeptide (M. Uhlen et al., Methods Enzymol., 185:129 (1990)). For example, the purification can be carried out by affinity chromatography (F. H.
  • the fractions containing the polypeptide of the present invention can be detected by EIA or western analysis using antibodies reactive with the polypeptide.
  • the antibodies reactive with the polypeptide of the present invention can be obtained by synthesizing the oligopeptide corresponding to the N-terminal 24-30 amino acid residues, conjugating it with carrier proteins such as bovine serum albumin and KLH (keyhole lymphet hemocyanin), and immunizing rabbits or other animals using a standard method (E. Harlow and D. Lane, “Antibodies”, Cold Spring Harbor Laboratory Press, 1988). It is also possible to obtain the antibodies reactive with the polypeptide of the present invention by producing in E. coli a fusion protein of the polypeptide of the present invention and another polypeptide, purifying the fusion protein by taking advantage of the partner polypeptide's properties, and by using it as the immunogen.
  • the polypeptide of the present invention binds to VEGF, this activity can be used as an index for the purification process.
  • a solution containing the non-purified polypeptide of the present invention is appropriately diluted and a 96-well polystyrene microtiter plate is coated with the solution, followed by blocking in the same manner as in preparing a antibody-coated plate for EIA. Since the thus-obtained plate specifically binds to VEGF, the binding can be detected by measuring the residual radioactivity in the wells using the 125 I-labeled VEGF.
  • a fraction from the chromatography used for purifying the polypeptide of the present invention is preincubated with 125 I-VEGF and the mixture is placed into the wells of the plate to measure the residual radioactivity. If the fraction contains the polypeptide of the present invention, its presence can be confirmed because it will bind to VEGF during the preincubation, which will cause a competition with the polypeptide of the present invention on the surface of the plate, thereby reducing the binding of VEGF to the plate.
  • FIG. 1 schematically shows the constitution of the extracellular domain of FLT.
  • FIG. 2 schematically shows the process of constructing a recombinant baculovirus that expresses 4N-FLT.
  • FIG. 3 schematically shows the process of constructing a recombinant baculovirus that expresses 5N-FLT.
  • FIG. 4 shows the results of the western blotting using the culture supernatants and the extracts of the cells infected with the recombinant B4N or B5N baculovirus.
  • FIGS. 5A and 5B show the Scatchard analyses of the interactions between 4N-FLT or 5N-FLT and the VEGF fragment.
  • FIG. 6 shows the inhibition of the VEGF-dependent thymidine uptake in HUVEC by the culture supernatants of EDF-, 4N-FLT-, or EDF ⁇ 11-expressing cells.
  • FIGS. 7A and 7B show the autoradiography following the electrophoresis of the covalently cross-linked products between the cells infected with an EDF- or an EDF ⁇ 11-expressing recombinant baculovirus and 125 I-VEGF 121 .
  • FIGS. 8A and 8B show the Scatchard analyses of the interactions between EDF or EDF ⁇ 11 and the VEGF fragment.
  • FIGS. 9A and 9B show the VEGF-affinity chromatogram and the electrophoresis pattern of each fraction during the EDF ⁇ 11 purification process.
  • FIGS. 10A and 10B show the electrophoresis pattern and the western analysis of the purified EDF ⁇ 11.
  • FIG. 11 shows a schematic diagram of the Fc domain of human immunoglobulin IgG 1 and the relative positions of the primers used for isolating fragments.
  • FIG. 12 shows the agarose gel electrophoresis patterns of the PCR-amplified and purified DNA and the recombinant plasmid digested with restriction enzymes.
  • FIG. 13 shows the agarose gel electrophoresis patterns of the PCR-amplified and purified DNA.
  • FIG. 14 schematically shows the process of constructing the recombinant FLT immunoglobulin-like domains 1&2-human IgG 1 -Fc expression plasmid, starting from the isolation of each DNA fragment.
  • FIG. 15 shows the western blot of the recombinant E. coli crude extract by the anti-human IgG 1 -Fc monoclonal antibody.
  • FIG. 16 shows the SDS polyacrylamide gel electrophoresis patterns of the covalently cross-linked products between the recombinant E. coli crude extract and 125 I-VEGF 165 .
  • FIG. 17 shows the Scatchard analyses of the binding between the FLT immunoglobulin-like domains 1&2-human IgG 1 -Fc fusion protein in the recombinant E. coli crude extract and 125 I-VEGF 165 .
  • FIG. 18 shows the VEGF inhibitory activity of the partially purified FLT immunoglobulin-like domains 1&2-human IgG 1 -Fc fusion protein.
  • Plasmid pflt3-7 (M. Shibuya et al., Oncogene, 5:519 (1990)) DNA was digested with restriction enzymes EcoRI and NdeI. and an EcoRI-NdeI DNA fragment of approximately 1.6 kbp was prepared following separation by agarose gel electrophoresis.
  • the plasmid pME18Sneo DNA was digested with restriction enzymes EcoRI and XhoI, and an EcoRI-XhoI DNA fragment of approximately 5.5 kbp was prepared following separation by agarose gel electrophoresis.
  • the oligonucleotides having the following sequences “5′ TATTAATGATCTAGATGAC 3′” (SEQ ID NO:4) and “5′ TCGAGTCATTCTAGATCATTAA 3′” (SEQ ID NO:5), were mixed as adapters at the room temperature, the above “1.6 kbp EcoRI-NdeI DNA fragment” and “5.5 kbp EcoRI-XhoI DNA fragment” were added thereto. Then, ligation was carried out using T4 DNA ligase and the resulting DNA was introduced into E. coli . The resulting plasmid DNA was digested with EcoRI and NotI.
  • Construction of a vector was carried out following the process as shown in FIG. 3 according to the method described in “Molecular Cloning” by J. Sambrook et al.
  • the plasmid pflt3-7 DNA was digested with EcoRI and HindIII, and a 1.9 kbp EcoRI-HindIII DNA fragment was prepared.
  • the oligonucleotides having the following sequences “5′ AGCTTTTAATGATCTAGAATGAC 3′” (SEQ ID NO:6) and “5′ TCGAGTCATTCTAGATCATTAAA 3′” (SEQ ID NO:7), were mixed as adapters and added to the above fragment to ligate with the 5.5 kbp EcoRI-XhoI DNA fragment from the plasmid pME18Sneo described above. The resulting ligation product was used to transform E. coli cells to give a recombinant plasmid.
  • the resulting plasmid DNA was digested with EcoRI and NotI, and the 1.9 kbp fragment thus produced was recovered and inserted into the EcoRI/NotI sites of pVL1393 (PharMingen).
  • This plasmid DNA was purified and used to construct the recombinant virus using BaculoGold (PharMingen), which is a baculovirus DNA lacking the Polyhedrin coding region, according to the manual.
  • BaculoGold BaculoGold
  • This recombinant baculovirus was named “B5N.”
  • the recombinant baculovirus “B5N” was amplified in Sf9 cells according to the manual and used in the subsequent experiments. It should be noted that “B5N” contains the DNA encoding the amino acid sequence from the amino-terminus up to the 560th amino acid residue of FLT.
  • HIFiveTM insect cells (manufactured by Invitrogen Corp.) were cultured in the EXCELL 400TM culture medium (manufactured by Iwaki Glass) and infected with the recombinant baculovirus “B4N” or “B5N”. The culture supernatants were recovered and electrophoresed on an SDS-polyacrylamide gel, followed by western blotting.
  • a rabbit anti-FLT extracellular domain polyclonal antibody was used as the primary antibody and the alkaline phosphatase-labeled anti-rabbit IgG as the secondary antibody to develop color by adding NTB (Nitroblue tetazolium chloride)/BCI P (5-Bromo-4-chloro-3-indolylphosphate p-toluidine salt) (manufactured by Gibco BRL). The results indicated specific reactivity with this antibody (FIG. 4 ).
  • Lane 1 stands for the electrophoretic pattern of 2 ⁇ l of the culture supernatant of B4N-infected cells, lane 2 for 10 ⁇ l of the extract of B4N-infected cells, lane 3 for 2 ⁇ l of the culture supernatant of B5N-infected cells, and lane 4 for 10 ⁇ l of the extract of B5N-infected cells.
  • the mobility of the bands exhibiting the immunochemical reactivity was in agreement with their expected molecular weights.
  • these products were called “4N-FLT” and “5N-FLT,” respectively. It was estimated that the culture supernatant samples contained approximately 2 ⁇ g/ml of “4N-FLT” or “5N-FLT.”
  • HIFiveTM cells were infected with the recombinant virus “B4N” or “B5N”.
  • the recovered supernatants were diluted 4 times with PBS, 100 ⁇ l portions of which were dispensed into each well of a microtiter plate (“Immuron 2” manufactured by Dynatech). After the plate was kept overnight at 4° C., the liquid was removed from the wells and the wells were washed 3 times with PBS-0.1% BSA. Then, 250 ⁇ l of PBS-1% BSA was added thereto and the plate was left at room temperature for 2 hr for blocking.
  • the wells were emptied followed by adding 100 ⁇ l of a solution prepared by mixing 20,280 cpm of the 125 I-VEGF 165 (a peptide corresponding to the amino-terminus and up to the 165th residue of VEGF/manufactured by Amersham) having specific activity of 66,000 cpm/ng with 0-15,000 pg of unlabeled VEGF 165 .
  • the plate was left at room temperature for 3 hr.
  • the wells were then emptied and washed 3 times with PBS-0.1% BSA.
  • the residual radioactivity in the wells was measured by a Acounter for Scatchard analysis (FIG. 5 ).
  • 5A and 5B correspond to the results from the plates coated with the “B4N” and “B5N” expression culture supernatants, respectively. Since 125 I-VEGF 165 hardly binds to the plate coated with the culture supernatant from the control virus-infected Sf9 cells, the bound radioactivity can be considered to result from the binding of 125 I-VEGF 165 to the inserted gene expression products.
  • the affinity for VEGF 165 of the expression products in the culture supernatants of the “B4N”- or “B5N”- infected cells was calculated in Kd (dissociation constant) of approximately 3-4.5 ⁇ 10 ⁇ 11 , which was close to the values reported for FLT (J. Waltenberger et al., J. Biol. Chem., 269:26988 (1994)) or soluble FLT (R. L. Kendal and K. A. Thomas, Proc. Natl. Acad. Sci. U.S.A., 90:10705 (1993)).
  • the inhibitory effect of “4N-FLT” or “5N-FLT” on VEGF-dependent proliferation was examined.
  • the sinusoidal endothelial cells were prepared from the rat liver according to the method as described and inoculated at 10 4 cells/well onto 24-well plates. The cells were cultured for 4 days in the presence of the samples listed in Table 2 and the cells in each well were counted. The numerals in the column of the cell number in Table 2 stand for relative values with taking the number of cells immediately after the inoculation as 100.
  • the corresponding recombinant virus-infected HiFive culture supernatants were used and the concentrations were calculated based on the results of the western analysis. The results indicate that “4N-FLT” and “5N-FLT” inhibit the VEGF's endothelial cell proliferation activity dose-dependently.
  • VEGF-dependent proliferation inhibition activity of “4N-LFT” and “5N-LFT” on rat liver sinusoidal endothelial cells VEGF 4N-FLT 5N-FLT concentration concentration concentration (ng/ml) (ng/ml) (ng/ml) Cell number 0 0 0 0 1 0 0 260 1 40 0 40 1 100 0 44 1 0 40 27 1 0 100 28 3 0 0 300 3 40 0 191 3 100 0 69 3 0 40 21 3 0 100 83
  • RNA was obtained.
  • 1 ⁇ l of 10% SDS was added 1 ⁇ l of 10% SDS and 100 ⁇ l of “Oligotex-dT30 (manufactured Takara Shuzo)”.
  • the resulting mixture was incubated at 65° C. for 5 min and then rapidly cooled in ice.
  • This solution was mixed with 20 ⁇ l of 5 M sodium chloride and incubated at 37° C. for 10 min.
  • the suspension thus obtained was centrifuged at 15,000 rpm for 15 min to recover the precipitate, which was resuspended in 100 ⁇ l of heat-sterilized pure water and incubated at 65° C.
  • HUVEC poly(A) + RNA 100 ⁇ l of the HUVEC double-stranded cDNA solution primed by oligo dT was obtained using this solution and the cDNA synthesis kit manufactured by Pharmacia following the manual.
  • PCR was performed with the following conditions.
  • the primer sequences are as follows:
  • Primer 1 5′-CTCGGATCCGGA TCTAGTTCAGGTTCAAAA -3′ (SEQ ID NO:8)
  • Primer 2 5′-CTCGAATTCA CTCCAGATTAGACTTGTCCGA -3′ (SEQ ID NO:9)
  • the recovered DNA was digested with HincII, HindIII, HhaI, or PstI to confirm the digestion patterns were in agreement with the patterns expected from the nucleotide sequence of the FLT extracellular domain encoding DNA.
  • This DNA fragment was then digested with EcoRI and BamHI, the resulting reaction mixture was treated with an equal volume of TE-saturated phenol, and the EcoRI, BamHI-digester DNA fragment was recovered from the aqueous layer using a Prep-A-GeneTM Kit.
  • plasmid A vector pGEX2T (manufactured by Pharmacia Biosystem) was digested with EcoRI and BamHI, the resulting reaction mixture was treated with an equal volume of TE-saturated phenol, and the EcoRI, BamHI-digested pGEX2T DNA was recovered from the aqueous layer using “Prep-A-Gene.”
  • the DNA fragment and the plasmid DNA thus obtained were mixed at a molar ratio of 10:1 and ligation was performed (Ligation Kit manufactured by Takara Shuzo).
  • E. coli JM109 competent cells manufactured by Takara Shuzo
  • the 2 ⁇ TY culture medium trypton 16 g, yeast extract 10 g, sodium chloride 5 g, and agar 1.5 g per 1 liter
  • agar 1.5 g per 1 liter containing 75 ⁇ g/ml ampicillin
  • the ampicillin-resistant colonies that emerged on the plate were picked up with toothpicks, transferred into 15 ⁇ l of the PCR reaction mixture which was identical to the above-described one except for lacking the template to perform PCR as described above for 30 cycles.
  • the reaction mixture was subjected to agarose gel electrophoresis, single colonies were isolated from the ones that produced a 2.2 kbp band and the plasmid DNA was prepared from a small quantity of the culture medium (according to the procedures described in J. Sambrook et al., “Molecular Cloning,” Cold Spring Harbor Laboratory Press, 1989). These plasmid DNAs were digested with BamHI and EcoRI and it was confirmed that they generated a 2.2 kbp fragment.
  • the E. coli clone which contains the above plasmid whose partial nucleotide sequence was confirmed, was shake-cultured at 30° C. in 500 ml of the 2 ⁇ TY culture medium containing 50 ⁇ g/ml ampicillin. IPTG was added to give the final concentration of 0.1 mM when the absorbance at 600 nm reached 1.0 and the medium was further cultured for another 20 hr. The cells were recovered by centrifugation and about 7.1 mg of the GST-EDF fusion polypeptide. with a purity of approximately 60% was prepared using glutathione-Sepharose (Pharmacia Biosystem) according to the procedure described in the manual.
  • the fusion polypeptide had a molecular weight of 60,000, from which the molecular weight of the GST of 28,000 was subtracted to give the partner for the fusion polypeptide of the molecular weight of about 32,000. Since the binding ability of GST to glutathione-Sepharose suggested that the GST portion was hardly decomposed, the latter half of EDF was presumed to have been lost, leaving the N-terminal portion with the molecular weight of 32,000.
  • the GST-EDF fusion polypeptide prepared in 3) above was mixed with Freund's complete adjuvant and subcutaneously injected to rabbits in a dose of initially 200 ⁇ g and subsequently 100 ⁇ g every other week per animal for a total of seven times.
  • a measurement of titer using antigen-coated plates revealed that the sera possessed sufficient immunological reactivity after more than 64,000-fold dilution.
  • 150 mg of the IgG fraction was obtained from the sera of two rabbits according to the procedures described in “Antibodies” by E. Harlow and D. Lane.
  • PCR was performed with the plasmid pflt3-7 (M. Shibuya et al., Oncogene, 5:519 (1990)) as the template under the following conditions.
  • Primer 3 5′-TTTCTCGGATCCTATAAAT ATGGTCAGCTACTGGGACACC -3′ (SEQ ID NO:10)
  • Primer 4 5′-GTGGTGGTGGTGGTGGTGACG CTCCAGATTAGACTTGTCCGA -3′ (SEQ ID NO:11)
  • PCR reaction was performed under the following conditions using the plasmid pRc/RSV (manufactured by Invitrogen Corp.) as the template to give a DNA (0.3 kbp) containing the polyadenylation signal derived from the bovine growth hormone gene.
  • Primer 5 5′-CACCACCACCACCACCACCACTAACTAGAGCTCGCTGATC-3′ (SEQ ID NO:12)
  • Primer 6 5′-TTCTCGAATTCTCCCCAGCATGCCTGC-3′ (SEQ ID NO:13)
  • a 200 ⁇ l portion of each reaction mixture thus obtained was treated with an equal volume of chloroform and the aqueous layer was recovered. Reagents were added thereto to make their final concentrations be 0.5% for SDS, 0.1 M for Tris-HCl (pH 6.8), 5 mM for EDTA, and 200 ⁇ g/ml for proteinase K. The solutions were incubated at 37° C. for 30 min. These solutions were treated with TE-saturated phenol, the aqueous layers were subjected to ethanol precipitation, and the DNA fragment encoding EDF and the DNA containing the polyadenylation signal were dissolved in TE.
  • the DNA solution was subjected to agarose gel electrophoresis to recover the 2.5 kbp DNA fragment considered to be the fusion product between the DNA fragment encoding EDF and the DNA fragment containing the polyadenylation signal, which was dissolved in 50 ⁇ l of a TE solution.
  • a DNA fragment was prepared by digesting the both ends of this DNA fragment with BamHI and EcoRI.
  • 1 ⁇ g of the plasmid pVL1393 (PharMingen) which is a transfer vector for the recombinant baculovirus, was digested with BamHI and EcoRI to prepare the BamHI-EcoRI fragment following the method described in the Example 5-2).
  • This plasmid DNA was mixed with the DNA fragment encoding EDF, which was obtained by the PCR amplification of plasmid pflt3-7 followed digestion with BamHI and EcoRI, at a molar ratio of approximately 1:5 and treated with the Ligation Kit (manufactured by Takara Shuzo).
  • the ligation product was used to transform competent E. coli JM109 cells and six clones containing the recombinant plasmid were selected using a method similar to the one described in Example 5-2).
  • Plasmid DNA was prepared from a 3 ml culture of each clone by the alkali method and the nucleotide sequence was determined for about 300 bp upstream and about 500 bp downstream of the inserted fragment.
  • the plasmids derived from two different kinds of clones had the correct sequences. These plasmids were designated “pEDFH10” and “pEDFH11.”
  • the E. coli cells harboring these plasmids were cultured in 100 ml of the 2 ⁇ TY medium containing 50 ⁇ g/ml ampicillin overnight at 37° C.
  • the plasmid DNAs were extracted from the recovered cell bodies using the alkali method (according to J.
  • Sf9 cells (manufactured by Invitrogen Corp.) cultured in the TMN-FH medium (manufactured by PharMingen) at an 80% confluency were detached by pipetting, inoculated at 2 ⁇ 10 6 cells per 60 mm dish, and allowed to adsorb onto the surface by standing for 30 min. Then, the medium was replaced with 2 ml of Ex-Cell 400 (manufactured by Iwaki Glass), which is a serum-free medium.
  • TMN-FH medium manufactured by PharMingen
  • the media were recovered and the supernatants obtained by centrifugation were prepared as the original virus stocks (designated “BEDFH10” and “BEDFH11” respectively).
  • the plaque assay performed according to the manual of Invitrogen Corp. revealed that both of these viruses had a titer of approximately 3 ⁇ 10 6 .
  • two clones (“BEDFH101” and “BEDFH102” from “BEDFH10” and “BEDFH111” and “BEDFH112” from “BEDFH11) were obtained by plaque isolation followed by 4-step amplifications according to the manual of Invitrogen Corp. to give about 200 ml of the virus solution (having a titer of about 5 ⁇ 10 7 /ml).
  • Sf9 cells were infected with the recombinant virus “BEDFH10” or “BEDFH11” at m.o.i. of 5 and 100 ⁇ l of the culture supernatants after 7 days of culturing were placed in the wells of microtiter plates (Immuron manufactured by Dynatech), followed by incubation at 4° C. overnight. The wells were emptied and washed 3 times with PBS-0.1% BSA. Then, 250 ⁇ l of PBS-1% BSA was added thereto and allowed to stand at room temperature for 2 hr for blocking.
  • FIGS. 8A and B show the results with the plates coated with EDF- and “EDF ⁇ 11”-expressing culture supernatants, respectively.
  • 125 I-VEGF 165 was hardly bound to the plate coated with the culture supernatant of the control virus-infected Sf9 cells, the bound radioactivity can be considered to represent the binding of 125I-VEGF 165 to the inserted gene expressed products.
  • Nucleotide sequences were determined in order to confirm the cloned DNA's sequences in the plasmids “pEDFH10” and “pEDFH11” which revealed that the nucleotide sequence of the part of the insert DNA in “pEDFH10”, which corresponds to the FLT extracellular domain, matched completely with that of FLT.
  • the nucleotide sequence of the part of the insert DNA in “pEDFH11”, which corresponds to the FLT extracellular domain lacked the C at nucleotide position 1053 of SEQ ID NO:1, as compared with FLT.
  • the open reading frame corresponded to residues-22 to 246 of the FLT amino acid sequence of SEQ ID NO:1. This portion contains the first and second domains of FLT.
  • Ni ++ -NTA (QIAGEN, Diagen GimbH) equilibrated with 20 mM Tris-HCl (pH 7.8)/50 mM KCl/0.1% Nonidet P-40/1 mM imidazole-HCl (pH 7.8) was added thereto and mixed at 4° C. for 1 hr. After centrifugation at 10,000 rpm, 30 ml of 150 mM KCl/0.1% Nonidet P-40/40 mM imidazole-HCl (pH 7.8) was added to the precipitate and mixed at 4° C. for 15 min. The precipitate was recovered by centrifugation and washed with the same buffer two more times.
  • the column was washed with 0.1 M phosphate buffer (pH 7.0)—0.3 M NaCl until the absorbance at 280 nm became sufficiently low and eluted with a 0.3 M-1.0 M linear gradient of NaCl. The fractions absorbing at 280 nm were recovered and VEGF affinity chromatography was performed.
  • the sample was diluted 2-fold with PBS, and loaded on 0.4 ml of the Sepharose 4B column coupled with 1.4 mg VEGF.
  • the column was washed with 20 ml PBS-0.5 M NaCl, and eluted with 10 mM sodium acetate into the tube containing 0.05 ml 2M Tris-HCl (pH 8.0) at 0.5 ml/tube.
  • each fraction was subjected to SDS-polyacrylamide gel electrophoresis according to Laemmli, and silver-stained (FIG. 9A; lane numbers correspond to fraction numbers).
  • Each fraction was also diluted 10 times with PBS-0.1% BSA, mixed with an equal volume of 125 I-VEGF 165 , and 1 hr thereafter, the VEGF binding was examined with 100 ⁇ l of the mixture using the microtiter plate used in Example 3-2).
  • the fractions showing a band at the molecular weight of 35,000 by electrophoresis which was almost in agreement with the value estimated from the results of the covalent cross-linking experiment, exhibited inhibition of VEGF binding (FIG. 9 B).
  • EDF or “EDF ⁇ 11” was subjected to SDS-polyacrylamide gel electrophoresis and western blotting was performed according to the method described in “Antibodies” by E. Harlow and D. Lane.
  • the antibodies obtained in Example 1-4) were used at 2 ⁇ g/ml as the primary antibody, and a 5,000-fold diluted alkaline phosphatase-labeled anti-rabbit IgG (E. Y. Laboratories) as the secondary antibody to develop color by adding NTB (Nitroblue tetazolium chloride)/BCIP (5-bromo-4-chloro-3-indolylphosphate p-toluidine salt) (manufactured by Gibco BRL).
  • NTB Nonroblue tetazolium chloride
  • BCIP 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt
  • FIG. 10A lane 1 represents the sample after the heparin column and lane 2 after the VEGF affinity chromatography, both electrophoresed and silver-stained.
  • FIG. 10B represents western blotting of the same samples.
  • HUVEC cells human umbilical cord-derived vascular endothelial cells (HUVEC).
  • HUVEC cells manufactured by Kurabo
  • a 96-well collagen plate manufactured by Iwaki Glass
  • 3,000 cells/well/100 ⁇ l EMM-UV medium manufactured by Kurabo
  • the cells were washed twice with PBS, 50 ⁇ l of 20 ng/ml VEGF 165 and 50 ⁇ l of the sample were added thereto followed by culturing for 4 days. Ten ⁇ l of 50 ⁇ Ci/2 nmoles/ml of 3 H-thymidine was added to each well and incubated for another 24 hr. After washing twice with PBS, the cells were detached by trypsin/EDTA and recovered on a glass filter by Cell Harvester (manufactured by Cambridge Technology, Inc.) to measure radioactivity with a scintillation counter (FIG. 6 ).
  • the recombinant virus-derived “EDF,” “4N-FLT,” or “EDF ⁇ 11”-expression culture supernatant showed a significant inhibition of the VEGF-dependent thymidine uptake.
  • a IgG-producing cell line human lymphoblastoma IM9 (Dainippon Pharmaceuticals), was cultured in the RPMI 1640 medium and the supernatant was examined using the Human IgG subclass profile kit (Zymed). As a result, the cell line was found to be producing human IgG 1 .
  • a cDNA solution was prepared from 4 ⁇ 10 7 IM9 cells by the same method as described in Example 5-1) of II. From this cDNA, the human IgG 1 -Fc cDNA fragment was amplified in two steps of PCR using the conditions listed in Table 7 below.
  • Primer 7 5′-TCTTGTGACAAAACTCACACATGC-3′ (SEQ ID NO:14)
  • Primer 8 5′-CGGAGACAGGGAGAGGCTCTTCTG-3′ (SEQ ID NO:15)
  • Primer 9 5′-GAGCCCAAATCTTGTGACAAAA-3′ (SEQ ID NO:16)
  • Primer 10 5′-TTCTC GGATCC TTA TTTACCCGGAGACAGGGA -3′ (SEQ ID NO:17)
  • Primer 10 indicates the restriction enzyme BamHI recognition site, “STP” the stop codon, and “hIgG1-Fc term” a C-terminal portion of the human IgG 1 -Fc coding region. Note that Primer 8 and Primer 10 are antisense strands. The relationships between the human IgG 1 -Fc coding region and the positions of the primers are shown in FIG. 11 .
  • the PCR reaction mixture was treated as in Example 5-2) of II and subjected to agarose gel electrophoresis. A DNA fragment of approximately 700 bp was excised and the DNA was recovered by centrifugation in a SUPEREC-01 filter tube (Takara Shuzo).
  • the HB101 strain of E. coli was cultured in 2 ⁇ TY medium at 37° C., overnight, and the cell body was recovered by centrifugation and resuspended in 0.5 ml TE buffer (10 mM Tris-HCl (pH 7.5)/1 mM EDTA). After lysis by adding 25 ⁇ l of 20 mg/ml egg white lysozyme and incubating at room temperature for 15 min, 50 ⁇ l of 10% SDS and 0.5 ml TE-saturated phenol were added thereto and vigorously shaken for 5 min. The aqueous layer was recovered by centrifugation and treated with an equal volume of chloroform to remove the phenol.
  • the DNA was precipitated by adding a double volume of ethanol and washed with 70% ethanol followed by drying. The resulting precipitate was dissolved in 200 ⁇ l of 20 ⁇ g/ml RNase A solution to prepare an E. coli genome DNA solution. This was used as the template to perform PCR according to the conditions given in Table 9 to provide an amplified Omp A signal peptide encoding DNA.
  • Primer 11 5′- TAACCTGGCG ATAAC GAGG CGCAAATA ATGAAAAAG -3′ (SEQ ID NO:18) trx term SD omp init Primer 12 5′-CTGAACTAGA TTTCGGAGCGGCCTGCGCTA -3′ (SEQ ID NO:19) mflt omp SP term
  • trx term indicates the terminal coding sequence of the E. coli thioredoxin gene (trx A), “SD” the ribosome binding sequence of omp A, and “omp init” the vicinity of the initiation codon-containing sequence of omp A.
  • Concerning Primer 12 “mflt” indicates the N-terminal coding region of the mature FLT, and “omp SP term” the terminal portion of the Omp A signal peptide coding region. Note that Primer 12 is an antisense strand.
  • the sequence of the thioredoxin gene was based on “B. J. Wallace and S. R.
  • PCR was performed and the E. coli thioredoxin gene was amplified.
  • Primer 13 5′-TTCTC GAATTC CCTGT GGAG TTATAT ATG AGC-3′ (SEQ ID NO:20)
  • Eco SD trx init Primer 14: 5′-G CCTC GTTAT CGCCAGGTTAGCGTCGAGGA -3′ (SEQ ID NO:21) omp SD trx term
  • Primer 13 “Eco” indicates the restriction enzyme EcoRI recognition site, “SD” the ribosome binding sequence of the E. coli thioredoxin gene, and “trx init” the vicinity of the initiation codon-containing sequence of trx A.
  • EDF ⁇ 11-expressing baculovirus vector pEDFH11
  • PCR was performed under the conditions listed in Table 11, to amplify the DNA encoding the immunoglobulin-like domains 1 and 2 of FLT.
  • the reaction mixture was purified as described above, and used as the EDF12 DNA (FIG. 13 ).
  • Primer 15 5′- CGCTCCGAAA TCTAGTTCAGGTTCAAAATT -3′ (SEQ ID NO:22) omp SP term mFLT
  • Primer 16 5′- TTTgTCACAAgATTTgggCTCT gTgCTTATTTggACATCTAT -3′ (SEQ ID NO:23) hIgG-Fc hinge 214-FLT-208
  • hIgG-Fc hinge indicates the human IgG 1 hinge encoding DNA
  • 214-FLT-208 the DNA encoding amino acid residues 208 through 214 of FLT. Note that Primer 16 is an antisense strand.
  • the approximately 1.7 kbp DNA fragment thus obtained had an EcoRI and a BamHI restriction sequence, which was contained in Primer 10 or Primer 13, on either end.
  • This 1.7 kbp fusion DNA fragment was digested with EcoRI and BamHI.
  • an E. coli expression vector pTTQ18 manufactured by Amersham Japan was digested with EcoRI and BamHI.
  • These restriction enzyme digestion reaction mixtures were treated as in Example 5-2) to recover the DNAs.
  • the Ligation Kit (Takara Shuzo)
  • the ligation reaction was done at a vector: insert ratio of 3:1. Using this ligation mixture and E.
  • JM109 competent cells (manufactured by Takara Shuzo), the transformation was performed and the transformants viable on an agar medium containing ampicillin were selected. From these transformants, using the same method as in Example 5-2), several recombinant clones containing the 1.7 kbp insert DNA were obtained, one of which was named JM109 (pEDF12Fc).
  • lanes M, 1, 2, 3, 4, 5, and 6 represent the molecular weight standard, thioredoxin DNA, Omp A signal peptide DNA, EDF12Fc DNA, thioredoxin DNA-Omp A signal peptide DNA-EDF12Fc DNA fusion fragment, pEDF12Fc DNA digested with EcoRI+BamHI, and pTTQ18 DNA digested with EcoRI+BamHI. (Undigested plasmids are indicated by the circle.)
  • JM109 (pEDF12Fc) was shake-cultured at 37° C. in 70 ml of 2 ⁇ TY medium containing 100 ⁇ g/ml ampicillin, IPTG was added to 0.5 mM when the absorbance at 600 nm reached 1.0, and the medium was cultured for another 12 hr. After completion of the culturing, the culture medium was rapidly chilled, 70 ⁇ l of 33 ⁇ M pAPMSF (Wako Pure Chemical Industries) was added as a protease inhibitor followed by centrifugation to recover the bacterial cells.
  • pAPMSF Waako Pure Chemical Industries
  • the cells were resuspended in 5 ml of 10 mM Tris-HCl (pH 8.0)/33 nM pAPMSF, and more than 95% of the cells were disrupted by ultrasonication.
  • the suspension was centrifuged at 10,000 g for 20 min, the precipitate was dissolved in 5 ml of 0.1 M Tris-HCl (pH 8.0)/1 M KCl/33 nM pAPMSF/5 mM EDTA/1% Triton X100/0.1% Nonidet-P40, centrifuged at 20,000 g to remove the insoluble matters, and the supernatant was used in the following analyses as a crude extract.
  • a crude extract derived from JM109 (pTTQ18) containing only the vector portion was similarly prepared as a control.
  • VEGF 165 manufactured by R&D
  • VEGF 165 was diluted to 50 ng/ml with PBS and aliquoted at 100 ⁇ l/well on an Immuron 2 microtiter plate. After allowing to stand at 4° C. overnight, the plate was blocked as in Example 7-2).
  • JM109 (pTTQ18) and JM109 (pEDF12Fc) crude extracts were diluted 20-fold with PBS/0.1% BSA and aliquoted at 100 ⁇ l/well.
  • the plate was allowed to stand at room temperature for 1 hr and the wells were washed 6 times with PBS/0.1% BSA. Next, 100 ⁇ l of the peroxidase-labeled anti-human IgG antibody (#IM-0837 manufactured by MBL), diluted 1,000-fold with PBS/0.1% BSA, was added to each well allowed to stand at room temperature for 1 hr, followed by washing the well 6 times as described above.
  • the crude extract was subjected to western blot analysis using the method as in Example 7-5).
  • the crude extract was electrophoresed on an SDS-polyacrylamide gel according to Laemmli and electroblotted onto a PDVF membrane.
  • 0.4 ⁇ g/ml of an anti-human IgG 1 -Fc monoclonal antibody (MBL #IM-0280) was used as the primary antibody and a 4,000-fold diluted alkaline phosphatase-labeled anti-mouse IgG (Zymed) was used as the secondary antibody.
  • FIG. 15 the samples on lanes 1 and 2 were the JM109 (pTTQ18) crude extract and the JM109 (pEDF12Fc) crude extract, respectively. Since the expected recombinant protein consists of 446 amino acid residues, the size of the observed band was reasonable.
  • protein A-Sepharose column chromatography was performed, and the protein was eluted at pH 4.0.
  • BSA was added to the elution to 1 mg/ml and dialyzed against PBS to subject the resulting sample to the following experiments. The other samples were also dialyzed after an addition of BSA.
  • the recombinant protein of the present invention was examined for inhibitory activity against the VEGF-dependent enhancement of the 3 H-thymidine uptake of HUVEC.
  • Four ⁇ g/ml of human IgG 1 was used as a negative control and 2 ⁇ g/ml of purified EDF was used as a positive control.
  • the recombinant protein which was partially purified by protein A-Sepharose, was used at 1 ⁇ g/ml, a notable VEGF inhibitory activity was observed as with EDF (FIG. 18 ).
  • the addition of human IgG 1 did not result in such inhibition. From these results, it was demonstrated that the recombinant protein of the present invention could inhibit the vascular endothelial cell proliferation enhancement activity of VEGF.
  • the recombinant protein of the present invention had such biochemical characteristics of human IgG 1 -Fc as being recognized by the human IgG 1 -Fc-specific monoclonal antibody and as binding to protein A. Furthermore, it was demonstrated that the fusion protein of the present invention bound to VEGF with high affinity comparable to EDF and that it also inhibited the biological activity of VEGF.
  • the polypeptides of the present invention can be utilized in treating diseases accompanying pathological neovascularization, such as solid tumors, because they can inhibit the VEGF-stimulated neovascularization.
  • diseases accompanying pathological neovascularization such as solid tumors
  • they since they are constituted by human-derived amino acids, they are unlikely to trigger antibody production even if they are administered for a prolonged period of time.
  • they since they have smaller molecular weights than the conventional polypeptides (R. L. Kendal and K. A. Thomas, Proc. Natl. Acad. Sci. U.S.A., 90:10705 (1993)), it is easier to express them using recombinant DNA techniques, and they infiltrate into diseased sites more quickly.

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